Reagents for labeling biomolecules having aldehyde or ketone moieties

ABSTRACT

Novel fluorescent derivitization reagents are described that are suitable for coupling to biomolecules that contain aldehyde or ketone functional groups. In one embodiment is provided reagents that have the following formula: 
                 
 
wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R 5  is -L-Z; L is arylene, or a C 1-6  perfluoroalkylene, or a single covalent bond; Z is a carbonyl hydrazide, hydrazide, sulfonyl hydrazide, or a thiocarbonyl hydrazide; R 11 -R 14  are independently H, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alklyamino, di(C 2-12 alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo; and R 21 -R 24  are independently H, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alklyamino, di(C 2-12 -alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z. The method of treating a sample with the derivativization reagents is described. The reagents are particularly useful for labeling glycoproteins or glycopeptides, nucleic acids, and lipopolysaccharides in electrophoresis gels.

This Application claim benefit of U.S. Provisional 60/237,932, filedOct. 2, 2000.

FIELD OF THE INVENTION

This invention relates to fluorescent derivatization reagents that labelsubstances containing aldehydes, ketones, and similar functional groups,and their use in labeling glycoproteins and glycopeptides, nucleic acidsand lipopolysaccharides, and other biomolecules.

BACKGROUND

Fluorescent dyes are known to be particularly suitable for biologicalapplications in which a highly sensitive detection reagent is desirable.Fluorescent dyes are used to impart both visible color and fluorescenceto other materials. Particularly useful are fluorescent reagents thatexhibit selectivity in their labeling reactions, permitting thedetection and/or identification of particular substances, oridentification of characteristics of a sample.

A variety of detectable hydrazine, hydroxylamine and amine derivativeshave been described that share the utility of labeling aldehyde andketone functional groups. Among the most widely used of such reagentsare dansyl hydrazine, fluorescein thiosemicarbazide, various biotinhydrazides, biotin hydroxylamine (ARP), and various aromatic amines(2-aminopyridine, 8-aminonaphthalene-1,3,6-disulfonic acid,1-aminopyrene-3,6,8-trisulfonic acid, 2-aminoacridone) (as described byHaugland et al. MOLECULAR PROBES, INC. HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS, 7TH EDITION, on CD-ROM, Chapter 3, andreferences cited therein, which are incorporated by reference).

Most existing methods of labeling carbohydrates that utilize hydrazine,hydroxylamine and amine derivatization reagents have focused on labelingaldehydes present in, or introduced into, carbohydrates, particularlythe so-called “reducing sugars”. The adduct formed with the reducingsugar can be further stabilized by treatment with borohydride or acyanoborohydride. The derivatization reaction typically proceeds or isfollowed by a separation technique such as chromatography,electrophoresis, precipitation, affinity isolation or other means beforedirect or indirect detection of the labeled product.

In addition, some simple and complex sugars have been derivatized bycoupling to a carboxylic acid, as in glucuronides. Typically, theamine-, hydrazine- or hydroxylamine-substituted label functions as anucleophile, and the carboxylic acid must first be activated by formingan ester or an anhydride, using an activating agent such as acarbodiimide (for example ethyldimethylaminopropyl carbodiimide, orEDAC). Capillary electrophoresis has been particularly effective foranalysis of adducts or detectable derivatives with carbohydrates,including complex carbohydrates and carbohydrates obtained by hydrolysisof glycoproteins, gangliosides and other sugar-containing biomoleules.The detectable adducts thus formed in each case with the sugar orpolysaccharide can themselves be utilized as tracers, for receptorbinding, as enzyme substrates and for multiple other applications.

The derivatization reagents of this invention show fluorescenceproperties superior to those of any similar reagents that have beendescribed for detection of aldehyde- or ketone-containing molecules ingels, and permit bright fluorescent labeling of carbohydrates,glycoproteins, glycogen, lipopolysaccharides, and other aldehyde,ketone, carboxylic acid, or sulfonic acid containing substances. Unlikebiotin hydrazides, biotin hydroxylamines and digoxigenin hydrazides, allof which require blotting onto a membrane and the use of a secondarydetection reagent, the reagents of the invention permit rapid detectionof oxidized glycoproteins within gels.

The preferred reagents of the invention are well excited by ultravioletexcitation sources commonly used in epi-illuminators andtransilluminators. The dyes possess unusually high Stokes shifts ofgreater than about 150 nm, effectively reducing background due toautofluorescence and scattering of the exciting light. In addition, thestaining procedure of this invention is rapid and mild, exhibitssubstantially greater sensitivity than previously utilized labelingreagents, and employs a simple staining procedure.

A family of fluorogenic substrates that yield highly fluorescentproducts and that are related to the reagents of the invention has beenpreviously described (U.S. Pat. No. 5,316,906 to Haugland et al. (1994);U.S. Pat. No. 5,443,986 to Haugland et al. (1995); both incorporated byreference). However, these known fluorogenic substrates arenonfluorescent until enzyme action, are not substituted by a reactivefunctional group to label a desired target substance, and theirfluorescent products are designed to be insoluble under physiologicalconditions. In contrast, in preferred embodiments of the invention, thenon-fluorescent reagents only become fluorescent when bound to thebiomolecules with aldehyde or ketone moieties, e.g. in a gel or on amembrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Fluorescence excitation/emission spectra for Compound 5-labeledα1-acid glycoprotein. Approximately 7.5 μg of protein was loaded onto a13% SDS-polyacrylamide gel and subjected to electrophoresis by standardprocedures. After labeling with Compound 5 (as described in Example 19),the resulting fluorescent band was excised and placed in a 2 mm×10 mmquartz cuvette. The large Stoke's shift of Compound 5 is spectrallyseparable from the blue fluorescence emission of plastic backingscommonly used as supports for pre-cast polyacrylamide gels, and isspectrally separable from emission of red-fluorescent protein stains,allowing multicolor detection of glycosylated and unglycosylatedproteins in gels and on blots.

FIG. 2: Intensity profiles of a protein mixture separated by SDSpolyacrylamide gel electrophoresis and either labeled with Compound 5 orwith a nonspecific total protein stain (SYPRO RUBY protein gel stain).(A.) Labeling of glycoproteins with Compound 5. (B.) Staining ofproteins with SYPRO RUBY protein gel stain. The proteins evaluated are:A) α2-macroglobulin (180 kDa, glycosylated), B) phosphorylase b (97 kDa,nonglycosylated), C) glucose oxidase (82 kDa, glycosylated), D) bovineserum albumin (66 kDa, nonglycosylated), E) α1-acid glycoprotein (42kDa, glycosylated), F) carbonic anhydrase (29 kDa, nonglycosylated), G)avidin (8 kDa, glycosylated), H) lysozyme (14 kDa, nonglycosylated).Glycoproteins may be selectively labeled directly in gels using Compound5, with minimal nonspecific staining of nonglycosylated proteins.

FIG. 3: Sensitivity and linear dynamic range of glycoprotein detectionafter SDS-polyacrylamide gel electrophoresis and staining with Compound5. (A.) α1-acid glycoprotein (B.) avidin. α1-Acid glycoprotein is aglycoprotein containing 40% carbohydrate while avidin is a glycoproteincontaining 10% carbohydrate. 1 ng or less of each glycoprotein can bedetected and the linear dynamic range of detection exceeds 3 orders ofmagnitude. Conventional periodic acid Schiffs (PAS) detection using acidFuchsin sulfite permits detection of only 38 ng of glycoprotein with alinear dynamic range of only 30-fold.

FIG. 4: Mobility shift analysis of protein deglycosylation as detectedusing Compound 5: (A.) Line trace of the electrophoretic profile ofα1-acid glycoprotein prior to treatment with endoglycosidases. (B.) Linetrace of the electrophoretic profile of the same protein after exposureto endoglycosidases. Enzymatic removal of the carbohydrate residues fromthe glycoprotein results in a loss of staining using Compound 5, thusdemonstrating the specificity of detection for carbohydrates in thepresence of proteins.

FIG. 5: Intensity profile of electrophoretically separated Escherichiacoli serotype 055:B5 lipopolysaccharide detected with Compound 5. Thereagents of the invention represent a substantial improvement overpreviously published lipopolysaccharide staining methods, such as silverstaining or zinc-imidazole reverse staining of gels (requiring 200 to1000 ng of lipopolysaccharide) or digoxigenin hydrazide-basedimmunoblotting procedures (requiring 30 ng of lipopolysaccharide).

FIG. 6: Sensitivity and linear dynamic range of lipopolysaccharidedetection after SDS-polyacrylamide gel electrophoresis. Detection islinear from 2 ng to 4000 ng (R²=0.9901), exceeding the sensitivity ofmost common gel-based and blot-based methods of lipopolysaccharidedetection by 15-500 fold. The linear dynamic range of detection issimilarly improved over standard methods, extending over 3-orders ofmagnitude compared to the 10-30 fold range for other methods.

FIG. 7: Intensity profile of electrophoretically separated DNA molecularweight standards using Compound 5 (as described in Example 29). Lanesloaded with ribosomal RNA failed to label with Compound 5. Less than 1ng of acid-treated DNA could be detected, thus providing detectionsensitivity comparable to ethidium bromide. Unlike ethidium bromide,this procedure fails to detect even 500 ng of rRNA.

SUMMARY OF THE INVENTION AND DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention describes novel fluorescent derivatization reagentssuitable for coupling to target substances that contain aldehydes,ketones, carboxylic acids and sulfonic acids, thereby conferring theirchromophoric and fluorescence properties onto the conjugated substance.

The reagents of the invention are substituted by at least one reactivegroup (Z) that is a hydrazide or a hydroxylamine, that is bound to thearomatic portion of the reagent by a covalent linkage L.

The derivatization reagents of the invention have the formula:

where the substituents R¹ and R², when taken in combination, form afirst aromatic ring system. The first aromatic ring system comprises a5- or 6-membered aromatic ring that optionally incorporates one or moreheteroatoms N, O, or S. The first aromatic ring system optionallyincorporates 1 or 2 additional fused aromatic rings that each optionallyincorporate one or more heteroatoms. The first aromatic ring system isoptionally substituted by halogen, alkyl having 1-6 carbons,perfluoroalkyl having 1-6 carbons, alkoxy having 1-6 carbons, sulfo,carboxy, hydroxy, amino, alkylamino having 1-6 carbons, dialkylaminohaving 2-12 carbons, nitro, cyano, aryl, or any combination thereof, orthe first aromatic ring system is substituted by a covalently boundreactive group -L-Z. Typical nonhydrogen substituents on the firstaromatic ring system are halogen or alkoxy. Where the reagent issubstituted by halogen, it is typically substituted by F, Cl or Br.Typically, the first aromatic ring system is a benzene or naphthalenethat is optionally substituted as above. Preferably, the first aromaticring system is a benzene that is substituted once by -L-Z.

The substituents R³ and R⁴, when taken in combination, form a secondaromatic ring system. The second aromatic ring system comprises a 5- or6-membered aromatic ring that optionally incorporates 1-3 additionalheteroatoms N, O, or S, or oxo, thiooxo, sulfone, or aminofunctionalities. The second aromatic ring optionally incorporates 1 or 2additional fused aromatic rings that themselves optionally incorporateone or more heteroatoms. The second aromatic ring system is optionallysubstituted by halogen, alkyl having 1-6 carbons, perfluoroalkyl having1-6 carbons, alkoxy having 1-6 carbons, sulfo, carboxy, hydroxy, amino,alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons, nitro,cyano, aryl, or any combination thereof, or the second aromatic ringsystem is substituted by a covalently bound reactive group -L-Z. Typicalnonhydrogen substituents on the first aromatic ring system are halogenor alkoxy. Where the reagent is substituted by halogen, it is typicallysubstituted by F, Cl or Br. Typically the second aromatic ringincorporates two fused rings. In one aspect of the invention, the secondaromatic ring system is a quinazolinone, or a benzazole. In anotheraspect of the invention, the second aromatic ring system is abenzoxazole, a benzothiazole, or an indoline ring system.

In one embodiment, R² and R³, when taken in combination, form anadditional 5- or 6-membered ring that is doubly fused to the first andsecond aromatic ring systems. In this embodiment, the bridge connectingthe first ring system to the second ring system comprises a combinationof saturated or unsaturated carbon-carbon bonds, O, N, or S atoms. Thecarbon and nitrogen atoms of the bridging moiety are optionallysubstituted by alkyl having 1-6 carbons or by the reactive group -L-Z.

Preferably, the reagents of the invention comprise at least threearomatic rings, two of which are fused. Typically either the first ringsystem or the second ring system consists of two fused rings. Typically,the bridging moiety is not present.

The X moiety is OH or -NH-Q-R⁵, where the Q moiety is anelectron-withdrawing linking group. Any stable divalent radical that issufficiently electronegative is a suitable Q moiety. In one aspect ofthe invention, the Q moiety has a Hammett sigma constant more positivethan 0.20. In a particular aspect, the Q moiety is carbonyl (—(C═O)—),thiocarbonyl (—(C═S)—), sulfonyl (—(SO₂)—), or phosphoryl (—(PO₂)—). Ina preferred aspect of the invention, Q is carbonyl, thiocarbonyl orsulfonyl.

R⁵ is alkyl having 1-6 carbons, alkoxy having 1-6 carbons, alkylaminohaving 1-6 carbons, or R⁵ is the reactive group -L-Z. Preferably, X is—N-Q-R⁵ and R⁵ is -L-Z.

Z is a functional group capable of reacting with an aldehyde or ketoneto form a covalent bond. In one aspect of the invention, Z is analiphatic or aromatic amine, such as ethylenediamine, or an aminecovalently bound directly to the first or second aromatic ring system.In another embodiment Z is —NR⁶—NH₂ (hydrazide), —NR⁶(C═O)NR⁷NH₂(semicarbazide), —NR⁶(C═S)NR⁷NH₂ (thiosemicarbazide), —(C═O)NR⁶NH₂(carbonylhydrazide), —(C═S)NR⁶NH₂ (thiocarbonylhydrazide), —(SO₂)NR⁶NH₂(sulfonylhydrazide), —NR⁶NR₇(C═O)NR⁸NH₂ (carbazide), —NR⁶NR⁷(C═S)NR⁸NH₂(thiocarbazide), or —O—NH₂ (hydroxylamine), where each R⁶, R⁷, and R⁸ isindependently H, or alkyl having 1-6 carbons, preferably H. In oneaspect of the invention, Z is a hydrazide, hydroxylamine, carbohydrazideor a sulfonylhydrazide. Preferably, Z is a carbohydrazide or asulfonylhydrazide.

The covalent linkage L binds Z to the fluorophore, either directly (L isa single bond) or with a combination of stable chemical bonds,optionally including single, double, triple or aromatic carbon-carbonbonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds,carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, andphosphorus-nitrogen bonds. L typically includes ether, thioether,carboxamide, sulfonamide, urea, urethane or hydrazine moieties.

Preferred L moieties have 1-20 nonhydrogen atoms selected from the groupconsisting of C, N, O, P, and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Preferably L is acombination of single carbon-carbon bonds that optionally includes oneor two heteroatoms, or L comprises an aromatic ring. Examples of Linclude substituted or unsubstituted polymethylene, arylene,alkylarylene, arylenealkyl, or arylthio. In one embodiment, L contains1-6 carbon atoms; in another, L contains a thioether linkage.

Although the reagents of the invention may be substituted by more thanone -L-Z moiety, they must be substituted by at least one -L-Z moiety.Preferably, the reagent of the invention is substituted by only one -L-Zmoiety, and more preferably R⁵ is the only -L-Z moiety (bound to thereagent at Q).

The preferred reagents of the invention exhibit an excitation maximumgreater than about 250 nm, and a Stokes shift of the emission greaterthan about 100 nm, more preferably greater than about 150 nm

In one embodiment of the invention, the fluorophore has the structure

where W is (CH₃)₂C (isopropylidene), —CH=(methine), —CH₂—, S, O, or—(N—R⁹)— wherein R⁹ is H or alkyl having 1-6 carbons. J is —(C═O)—,—(SO₂)—, or —CH═; and n is 1 or 0. When W is —(N—R⁹)— and J is —(C═O)—,the products are quinazolinones (also referred to as quinazolones). WhenW is —(N—R⁹)— and J is absent (n=0), the product are benzimidazoles.When W is S and J is absent (n=0), the products are benzothiazoles. WhenW is O and J is absent (n=0), the products are benzoxazoles. When W andJ are each methine, the products are quinolines. When W isisopropylidene and J is absent (n=0), the products are indolines.

When the first aromatic ring and the second aromatic ring are both6-membered rings, and the bridging moiety forms a 5- or 6-membered ringbetween them, the products are phenanthridines. Typically, where thereagent of the invention is a phenanthridine, it has the structure:

In another aspect of the invention, the reagent of the invention is aquinazolinone, a benzimidazole, a benzoythiazole, a benzoxazole, aquinoline, an indoline, or a phenanthridine that is independentlysubstituted one or more times by F, Cl, Br, C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, C₁-C₆ alkoxy, nitro, cyano, or aryl, or any combinationsthereof.

In yet another aspect of the invention, the reagent of the invention isstructurally similar to a quinazolinone, a benzimidazole, abenzothiazole, a benzoxazole, a quinoline or an indoline but is furthermodified in that at least one of the aromatic rings is fused to at leastone additional aromatic ring that optionally incorporates at least onehetero atom N, O, or S.

For use in detecting glycoproteins or glycopeptides, nucleic acids, andlipopolysaccharides, the preferred reagent of the invention has theformula:

wherein Q is carbonyl, thiocarbonyl, or sulfonyl, preferably sulfonyl,and R⁵ is -L-Z. Typically, L is arylene (preferably phenylene), aperfluoroalkyl (preferably C₃₋₆), or a single covalent bond; and Z is acarbonyl hydrazide, hydrazide, sulfonyl hyrdrazide, or a thiocarbonylhydrazide. The substituents R¹¹-R¹⁴ are independently H, C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ perfluoroalkyl, C₁₋₆ alklyamino, C₂₋₁₂ dialkylamino,amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo.Typically, R¹¹-R¹⁴ are H, or only one of R¹¹-R¹⁴ is non-hydrogen, whichnon-hydrogen substituent is preferably F, Cl, Br, or alkoxy. Thesubstituents R²¹-R²⁴ are independently H, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆ alklyamino, C₂₋₁₂ dialkylamino, amino, carboxy,cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z. Typically,R²¹-R24 are H, or only one of R²¹-R24 is non-hydrogen (preferably R²²),which non-hydrogen substituent is preferably F, Cl, Br, sulfo, or L-Z.Reagent-Target Conjugates

The product of cross-coupling with a target substance that contains analdehyde, ketone, carboxylic acid, or sulfonic acid functionality is areagent-target conjugate. In the reagent-target conjugates, thefluorophore is bound to the target substance (S_(T)) by a covalentlinkage, L′.

The nature of the linkage, L′, is distinct from the linkage L thatoriginally bound the reactive Z moiety to the reagent of the invention.L′ typically comprises the original L linkage, and also incorporates theatoms originally present in the Z moiety and the functional groupconjugated thereto. Typical linkages with aldehydes or ketonesincorporate an oxime, an amide, a hydrazone, a carbohydrazone, athiocarbohydrazone, a sufonylhydrazone, a semicarbazone, athiosemicarbazone, or similar functionality reflecting the specificnature of the reactive group Z used and the aldehyde or ketoneconjugated therewith. Linkages with carboxylic acids are typicallyreferred to as carbohydrazides or as hydroxamic acids. Linkages withsulfonic acids are typically referred to as sulfonylhydrazides orN-sulfonylhydroxylamines. The linkage L′ may be formed completely by theconjugation reaction between the reagent and the target, or may besubsequently stabilized by chemical reduction.

The aldehyde or ketone functional group is typically naturally presenton the target substance prior to its conjugation to the reagent of theinvention. Alternatively, the aldehyde or ketone functionality is formedon the target substance by chemical, light, heat, radiation, orenzymatic treatment prior to reaction with a reagent of the invention.In one aspect of the invention, the target substance is treated with anoxidizing condition, such as a chemical oxidizing agent (for example aperiodate, a strong acid, or ozone), oxidizing radiation, photolysis, orenzymatic oxidation. Carboxylic and sulfonic acid functionalities aretypically already present in the target substance, but may requirespecial conditions for coupling to the reagents of the invention.

Although a variety of chemical oxidants such as dichromate andpermanganate are effective at oxidizing simple and complex alcohols toaldehydes or ketones, they can be harsh reagents when used withbiomolecules and may overoxidize alcohols to carboxylic acids, which donot couple without use of an auxiliary coupling agent such as EDAC. Morecommonly the oxidizing agent is one that oxidizes vicinal glycols oraminoalcohols. By far the most common reagent of this type is periodicacid (HIO₄) or one of its salts such as sodium periodate. Oxidation byperiodates is gentle and highly selective. The product(s) of thereaction typically contain two aldehyde moieties, although ketones arealso formed from appropriately substituted glycols. Some examples ofmolecules that contain periodate-oxidizable glycols that cansubsequently be derivatized by the reagents of the invention includemonosaccharides, disaccharides and polysaccharides; the 5′-terminalnucleotide in ribonucleic acids; serine, threonine and unblockedpeptides and proteins that contain terminal serine or threonineresidues; transfer ribonucleic acids loaded with serine or threonine;ethylene glycol, glycerol, glycerol 1-phosphate, 2-aminoalcohols andsimilar derivatives; many glycoproteins, including immunoglobulins,peroxidase, avidin; various glycolipids, glycosphingolipids,gangliosides and lipopolysaccharides; and numerous conjugates of drugs,ligands and natural products that contain periodate-oxidizable glycol oraminoalcohol functional groups.

An additional method for selective derivatization (and subsequentdetection) of certain carbohydrates, for example galactosides, isthrough the enzyme-catalyzed oxidation of the carbohydrate to analdehyde, which can be subsequently reacted with a detectable amine,hydrazine or hydroxylamine derivative. This treatment is primarilyeffective for carbohydrates in which catalytic oxidation yields analdehyde, such as galactose residues. Glucose oxidase, for instance,does not yield an aldehyde when it oxidizes glucosides. An applicationof this reaction is the highly selective modification and identificationof cell-surface galactoside-containing proteins in live cells usinggalactose oxidase.

Certain other molecules have intrinsic aldehydes or ketones that reactwith detectable amines, hydrazines or hydroxylamines spontaneously orwith moderate warming at an appropriate pH. In addition to reducingsugars and some oligosaccharides, in which the aldehyde (or ketone) ispredominantly in a ring structure, these reactions have frequently beenused for derivatization of low molecular weight molecules such as drugsand hormones (e.g. steroids) and other ligands that have molecularweights<2000, most often followed by separation from excess reagent andanalysis by a chromatographic technique such as thin layerchromatography, HPLC or electrophoresis. Only a few proteins, such aselastin, collagen, and substrates of lysyl oxidase, have intrinsicaldehyde or ketone functional groups, which permit their selectivedetection in the presence of other proteins without an oxidation step.Some additional natural products that can be derivatized include, butare not limited to, acetaldehyde, glyceraldehyde, dihydroxyacetone, andpyruvates.

An extremely wide variety of carboxylic acids are derivatized by thereagents of the invention, in conjunction with carboxylate-activatingreagents. Suitable carboxylic acids include C₁ to C₃₀ carboxylic acidsthat are linear, branched, saturated or unsaturated or that containadditional aliphatic or aromatic rings or additional substituents;intermediates in the citric acid cycle; synthetic polymers (includingcarboxylate-modified microspheres and membranes); amine-blocked aminoacids, peptides and proteins; drugs; ligands of MW<2000; and selectedpolysaccharides. Sulfonyl halides (expecially sulfonyl chlorides) arereadily coupled to amines, hydrazines and hydroxylamines of theinvention, to form stable sulfonamides.

The reagents of the invention can label nucleotides, oligonucleotidesand nucleic acids by multiple means, including 1) periodate oxidation ofthe 5′-terminus of ribonucleic acids followed by coupling to a Z moietythat is an amine or a hydrazine; 2) coupling to cytosine residuesmediated by bisulfite; 3) the Feulgen reaction, in which adeoxynucleoside, a deoxynucleotide, or an oligodeoxynucleotide of a DNAis treated with acid to yield a hemiacetal that is then coupled with areagent of the invention.

Abasic sites of nucleic acids (formed by radiation exposure, reactiveoxygen treatment and certain other chemical treatments), lack theirbases and comprise the same hemiacetal link that is formed by theFeulgen reaction. Thus these abasic sites are also detected with thereagents of the instant invention.

With the exception of some steroids, cellular lipids typically do notcontain aldehyde or ketone moieties. These are typically introduced byoxidation of alcohols or hydroperoxides. Carbon-carbon double bonds areconverted to glycols using a reagent such as osmium tetroxide, and arethen oxidized to aldehydes by a periodate. It is more common to detectlipid conjugates of sugars, such as lipopolysaccharides (LPS),gangliosides, cerebrosides and glycosylsphingolipids by periodateoxidation of their carbohydrate portion to aldehydes. The reagents ofthis invention provide exceptionally sensitive detection of oxidized LPS(Example 26).

Where they are not present, aldehydes and ketones are also introducedinto molecules using extrinsic reagents that already contain an aldehydeor ketone. For instance, aldehydes are introduced at aliphatic aminesites with the reagents succinimidyl 4-formylbenzoate or succinimidyl4-formylphenoxyacetate (Molecular Probes, Eugene OR). These reagentsselectively modify proteins on the surface of live cells, and therebypermit the analysis of the topology of peptide and protein exposure oncells surfaces following, for instance, lysis and gel electrophoresis.Additionally, galactosides are enzymatically transferred to a targetcarbohydrate using UDP-galactose:N-acetylglucosaminegalactosyltransferase and, following galactose oxidase-catalyzedoxidation to an aldehyde (as described by Shaper et al. J. SUPRAMOL.STRUCTURE 6, 291-299 (1977)), the target carbohydrate can be modified bya reagent of the invention. Glycoproteins such as horseradish peroxidaseare oxidized to aldehydes and their conjugates subsequently used invarious detection schemes according to the instant invention (Example28).

The oligosaccharide components of cell surface glycoproteins play a rolein the interactions that regulate many important biological processes,from cell-cell adhesion to signal transduction. Sialic acids are themost abundant terminal components of oligosaccharides on mammaliancell-surface glycoproteins and are synthesized from the six-carbonprecursor N-actylmannosamine. When cells in culture are incubated withN-levulinoyl-D-mannosamine, this ketone-containing monosaccharide servesas a substrate in the oligosaccharide synthesis pathway, resulting inketone-tagged cell-surface oligosaccharides (as described in U.S. Pat.No. 6,075,134 to Bertozzi et al. (2000), incorporated by reference). Ifthese tagged cells are then labeled with a reagent of the invention,they are readily identified or traced using either by imaging or flowcytometry.

The conjugated target is typically a peptide, a protein, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a sugar, apolysaccharide, a lipid, a lipopolysaccharide, a ganglioside, a drug, ahormone, or a ligand having a molecular weight less than 2,000 Daltons.Preferably, the conjugated target is a protein, a nucleic acid, a lipid,a lipopolysaccharide, a ganglioside, a drug, or a hormone. Mostpreferably, the conjugated target is a glycoprotein, alipopolysaccharide, or a nucleic acid.

Methods of Use

The use of the invention to label aldehyde- and ketone-containing targetsubstances comprises combining a reagent of the present invention with asample that contains or is thought to contain a desired target,incubating the mixture of reagent and sample for a time sufficient forthe reagent to form a covalent conjugate with the target substance inthe sample, such that the conjugate exhibits a detectable fluorescentsignal. Conjugation of the instant reagents with carboxylic acid- andsulfonic acid-containing substances requires prior activation of thesefunctional groups to an amine-reactive species.

The characteristics of the resulting reagent-target conjugate, includingthe presence, location, intensity, excitation and emission spectra,fluorescence polarization, fluorescence lifetime, photobleaching rateand other physical properties of the fluorescent signal can be used todetect, differentiate, sort, quantitate, and/or analyze aspects orportions of the sample. The reagents of the invention are optionallyused in conjunction with one or more additional detection reagents(preferably having detectably different fluorescence characteristics).

Selected Target Substances Containing Aldehydes or Ketones

-   Formaldehyde-   Acetone-   Benzaldehydes-   Reducing sugars and polysaccharides in ring-opened forms-   Steroids-   Keto acids-   Aldehyde- or ketone-containing drugs-   Aldehyde- or ketone-containing environmental pollutants-   Aldehyde- or ketone-containing organics-   Acid-treated deoxyribonucleic acids-   Oxidized sugars-   Oxidized polysaccharides-   Oxidized glycols-   Oxidized glycoproteins-   Oxidized glycolipids-   Oxidized glycosaminoglycans-   Oxidized ribonucleic acids-   Oxidized biological cells-   Oxidized N-terminal serine residues of proteins-   Oxidized N-terminal threonine residues of proteins    Selected Target Substances Containing Carboxylic Acids or Sulfonic    Acids-   Steroids-   Carboxylic acids-   carboxylic or sulfonic acid-containing drugs-   carboxylic or sulfonic acid-containing environmental pollutants-   carboxylic or sulfonic acid-containing organics-   fatty acids-   carboxylated sugars-   polymers-   biological cells-   proteins-   Staining Solution

Typically, when the reagent of the invention is used to detect aldehydesor ketones, it is used in the form of a staining solution, preferably anaqueous or aqueous miscible solution that is compatible with the sampleand the intended use. For biological samples, where minimal perturbationof cell morphology or physiology is desired, the staining solution isselected accordingly. For solution assays, the staining solutionpreferably does not perturb the native conformation of the targetsubstance.

Although typically used in an aqueous or aqueous miscible solution, thestaining solution is typically prepared by first dissolving the reagentin a water-miscible organic solvent such as dimethylsulfoxide (DMSO),dimethylformamide (DMF), or a lower alcohol, such as methanol orethanol. This stock solution is typically prepared at a concentration ofgreater than about 50-times that used in the final staining solution,then diluted one or more times with an aqueous solvent or a buffersolution such that the reagent is present in an effective amount.Typically, the reagent is first dissolved in 100% DMF, and then dilutedwith buffer. The staining solution optionally further comprisesadditional formulation components, such as acids, buffering agents,inorganic salts, polar organic solvents, antioxidants, and ionchelators.

The pH of the staining solution is optionally modified by the inclusionof a buffering agent. Any buffering agent that is compatible with thetarget substance in the sample is suitable for inclusion in the stainingsolution.

In one embodiment, the buffering agent is one of the so-called “Good's”buffers. “Good's” buffers include BES(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid;2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid), BICINE(N,N-bis[2-hydroxyethyl]glycine), CAPS(3-[cyclohexylamino]-1-propanesulfonic acid), EPPS(N-[2-hydroxyethyl]piperazine-N′-[3-propanesulfonic acid]), HEPES((N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), MES(2-[N-morpholino]ethanesulfonic acid), MOPS(3-[N-morpholino]propanesulfonic acid), PIPES(piperazine-N,N′-bis[2-ethanesulfonic acid];1,4-piperazinediethanesulfonic acid), TAPS (N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid; ([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino-1-propanesulfonic acid), TES(N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid;2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid), orTRICINE (N-tris [hydroxymethyl]methylglycine;N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine).

Other preferred buffering agents include salts of formate, citrate,acetate, N-(2-hydroxyethyl)-N′-(2-sulfoethyl)piperazine, imidazole,N-(2-hydroxyethylpiperazine)-N′-2-ethanesulfonic acid,Tris(hydroxymethyl)aminomethane acetate, or Tris(hydroxymethyl)aminomethane hydrochloride. In a preferred embodiment,the buffering agent is MES, sodium acetate, or acetic acid, preferablyacetic acid. The buffering agent or mixture of buffering agents istypically present in the staining solution at a concentration of 20 mMto 500 mM, preferably about 25 mM to about 100 mM. Where the bufferingagent is acetic acid, it is preferably present in a concentration ofabout 1%-6%, more preferably at about 3%.

In a particularly advantageous formulation of the staining solution, thestaining solution additionally comprises an inorganic salt. Advantageousinorganic salts produce staining formulations that exhibit lowbackground signals when staining glycoproteins in electrophoretic gels.Particularly useful and inexpensive salts include sodium chloride,ammonium sulfate, magnesium chloride, magnesium acetate, zinc chloride,magnesium sulfate and magnesium glucuronate present in the stainingsolution at a concentration of 1-50%. In a preferred embodiment, theinorganic salt is sodium chloride or magnesium chloride, more preferablymagnesium chloride.

An effective amount of reagent is the amount of reagent sufficient togive a detectable fluorescence response in combination with the desiredtarget. The reagent concentration in the solution must be sufficientboth to contact the target in the sample and to combine with the targetin an amount sufficient to give a signal, but too much reagent may causeproblems with background fluorescence or speckling in gels. The optimalconcentration and composition of the staining solution is determined bythe nature of the sample (including physical, biological, biochemicaland physiological properties), the nature of the reagent-targetinteraction (including the transport rate of the reagent to the site ofthe target), and the nature of the analysis being performed, and can bedetermined using standard procedures, similar to those described inexamples below.

Where the target substance contains a carboxylic acid or sulfonic acidfunctional group, the functional group must first be activated beforecombining with a staining solution containing a reagent of theinvention, depending upon the properties of the target substance.Typically carbodiimides, such as EDAC, or dicyclohexylcarbodiimide (DCC)are used to activate carboxylic acids, whereas sulfonic acids most oftenrequire formation of their sulfonyl chloride by standard means. Thereagent adducts of carboxylic acids and sulfonic acids are typicallyused to characterize the target substance, or the conjugates are used asfluorescent tracers. Carboxylic acid and sulfonic acids do not formstable adducts when stained in gels or solutions, thus differentiatingthem from aldehydes and ketones.

In one embodiment of the staining solution, the compound has the formulaof Formula XXX described above. In a preferred embodiment, the stainingsolution contains a compound selected from the group consisting ofCompounds 5, 20, 21, and 23 described below; more preferably Compounds5, 20, and 23.

Sample Types

The target of interest is optionally enclosed within a biologicalstructure (i.e. an organism or a discrete unit of an organism), free insolution (including solutions that contain biological structures),immobilized in or on a solid or semi-solid material, or is extractedfrom a biological structure (e.g. from lysed cells, tissues, organismsor organelles).

In one aspect of the invention, the target is a biological structure andis optionally a cell or tissue. Typically, the sample containing thedesired target is an aqueous or aqueous miscible solution that isobtained directly from a liquid source or as a wash from a solidmaterial (organic or inorganic) or a growth medium or a buffer solutionin which biological structures have been placed for evaluation.

In one aspect of the invention, the sample is obtained from a biologicalfluid, including separated or unfiltered biological fluids such asurine, cerebrospinal fluid, blood, lymph fluids, tissue homogenate,interstitial fluid, cell extracts, mucus, saliva, sputum, stool,physiological secretions or other similar fluids. Alternatively, thesample is obtained from an environmental source such as soil, water, orair; or from an industrial source such as taken from a waste stream, awater source, a supply line, or a production lot. Industrial sourcesalso include fermentation media, such as from a biological reactor orfood fermentation process such as brewing; or foodstuffs, such as meat,grain, produce, eggs, or dairy products.

In yet another embodiment, the sample is a solid or semi-solid matrixand the target of interest is present on or in the matrix. In one aspectof the invention, the matrix is a membrane. In another aspect, thematrix is an electrophoretic gel, such as is used for separation andcharacterization of proteins or nucleic acids. In another aspect, thematrix is a silicon chip, a glass fiber, or glass slide, and the targetof interest has been immobilized on the chip, fiber, or slide, such asin an array. In yet another aspect, the matrix is a polymericmicroparticle, and the target has been immobilized on the surface of themicroparticle.

The source and type of sample, as well as the use of the reagent, willdetermine which reagent characteristics, and thus which reagents, willbe most useful for staining a particular sample. Where the fluorescenceof the reagent-target complex or reagent conjugate is detected utilizingsustained high intensity illumination (e.g. microscopy), reagents with arate of photobleaching lower than commonly used reagents (e.g.fluorescein) are preferred, particularly for use in live cells. Wherethe reagent must penetrate cell membranes or a gel, more permeantreagents are preferred. Reagents that rapidly and readily penetrate cellmembranes do not necessarily rapidly penetrate gels.

In one aspect of the invention, the sample is also exposed to anoxidizing condition, such as a strong acid (the Feulgen reaction),oxidizing radiation, oxidizing enzymes, and chemical oxidizing agents.The sample is typically exposed to an oxidizing condition by combiningthe sample with an oxidizing agent. Typically the oxidizing agent isadded to the sample before combination with the staining solution. Inone aspect of the invention, the oxidizing agent is a depurinating agentthat generates an aldehyde on a nucleic acid. Preferably thedepurinating agent is HCl. In another aspect of the invention, theoxidizing agent is a periodate.

Formation of the Reagent-Target Complex

The sample is combined with the staining solution by any means thatfacilitates contact between the reagent and the target. The contact mayoccur through simple mixing, as in the case where the sample is asolution. Alternatively, the staining solution is added to the sampleand the resulting combined mixture is incubated for a time sufficient toform the reagent-target conjugate. A staining solution may contact thetarget in a liquid separation medium such as an electrophoretic liquid,sieving matrix or running buffer, or in a chemically compatiblesedimentation or buoyant density gradient (e.g. containing CsCl), or onan inert matrix, such as a blot or gel, a testing strip, or any othersolid or semi-solid support. Suitable supports also include, but are notlimited to, polymeric microparticles (including paramagneticmicroparticles), polyacrylamide and agarose gels, nitrocellulosefilters, optical fibers, computer chips (such as silicon chips), naturaland synthetic membranes, liposomes and chemically compatible hydrogels,and glass (including optical filters), and other silica-based andplastic support. The reagent is optionally combined with the sampleprior to undergoing gel or capillary electrophoresis, gradientcentrifugation, or other separation step, during separation, or afterthe sample undergoes separation. Alternatively, the reagent is combinedwith an inert matrix or solution in a capillary prior to addition of thesample, as in pre-cast gels, capillary electrophoresis or preformeddensity or sedimentation gradients. Alternatively, formation of thereagent-target conjugate occurs in a medium such as is used for anorganic synthesis.

Where the target is enclosed in a biological structure, the sample istypically incubated with the reagent. While some reagents may permeatebiological structures rapidly and completely upon addition of thereagent solution, any other technique that is suitable for transportingthe reagent into the biological structure is also a valid method ofcombining the sample with the subject reagent. Some cells activelytransport the reagents across cell membranes (e.g. endocytosis oringestion by an organism or other uptake mechanism) regardless of theircell membrane permeability. Suitable artificial means for transportingthe reagents across cell membranes include, but are not limited to,action of chemical agents such as detergents, enzymes or adenosinetriphosphate; receptor- or transport protein-mediated uptake; liposomesor alginate hydrogels; phagocytosis; pore-forming proteins;microinjection; electroporation; hypo-osmotic shock; or minimal physicaldisruption such as scrape loading, patch clamp methods, or bombardmentwith solid particles coated with or in the presence of the reagents.Preferably, where intact structures are desired, the methods forstaining cause minimal disruption of the viability of the cell andintegrity of cell or intracellular membranes. Alternatively, the cellsare fixed and treated with routine histochemical or cytochemicalprocedures, particularly where pathogenic organisms are suspected to bepresent. It should be well-understood that aldehyde fixatives such asacrolein or glutaraldehyde should be avoided, as even after fixationthey maintain a free aldehyde group that will cross-react with thereagents of the invention. However, the method of the invention iscompatible with paraffin-embedded tissues, and fixation schemes such as10% buffered formalin.

The sample is combined with the reagent for a time sufficient to formthe fluorescent reagent-target conjugate. Where the target is present ina gel or on a blot, sample is combined with the reagent for the timerequired to give a high signal-to-background ratio after washing.Optimal staining with a particular reagent is dependent upon thephysical and chemical nature of the individual sample and the samplemedium, as well as the property being assessed. The optimal time isusually the minimum time required for the reagent, in the concentrationbeing used, to achieve the highest target-specific signal while avoidingdegradation of the sample over time and minimizing all other fluorescentsignals due to the reagent. Where the reagent of the invention is usedsynthetically as a derivatization reagent, the optimal time for mixingis typically the minimum time required to form the highest yield of thecovalent adduct, as determined by experimentation. A preferred yield isa quantitative formation of the covalent adduct.

Preferably, when used with biological specimens, the reagent is combinedwith the sample at a temperature optimal for biological activity of thetarget within the operating parameters of the reagents (usually between0° C. and 60° C., with reduced stability of the reagents at highertemperatures). For in vitro assays, the reagent is typically combinedwith the sample at about room temperature (23° C.).

The method of the instant invention is useful for labeling targetmolecules in a variety of applications, including electrophoretic gels;thin layer chromatograms; targets present on blots, chips, strips andother surfaces; targets present in flowing systems, such as in flowcytometry, HPLC, capillary electrophoresis, and microfluidic devices;and in solution in multiwell plates, facilitating high throughputscreening by automated methods. In some cases the target substance islabeled with the reagent of the invention prior to a separationtechnique. Alternatively, target substances are first separated, as inelectrophoretic gels or on surfaces, prior to labeling with the reagentsof the invention.

Additional Reagents

The method of the present invention optionally further comprises one ormore additional reagents that are simultaneously or sequentiallycombined with the sample mixture, the staining solution, or the combinedmixture. An additional reagent is optionally a detection reagent thatcolocalizes with the target or other analyte to enhance the detectionthereof. The additional reagent is optionally selected to be useful foridentification of other components or characteristics of the samplemixture, such as a poly(amino acid) stain, nucleic acid stain, a stainfor lipids or carbohydrates, or a pH indicator. Alternatively, theadditional reagent is a detection reagent designed to interact with aspecific portion of the sample mixture, so as to probe for a specificcomponent of the sample mixture (such as an antibody or antibodyconjugate), where spatial coincidence of the reagent-target conjugateand the detection reagent indicates that the additional reagent is alsoassociated with the target.

The additional reagent also incorporates a means for producing adetectable response. A detectable response means a change in, oroccurrence of, a parameter in a test system that is capable of beingperceived, either by direct observation or instrumentally. Suchdetectable responses typically include the change in, or appearance of,color, fluorescence, reflectance, pH, chemiluminescence, infraredspectra, magnetic properties, radioactivity, light scattering, x-rayscattering, or the precipitation of an electron-rich product.Appropriate means to provide a detectable response include, but are notlimited to, a visible or fluorescent dye, a chemiluminescent reagent, anenzyme substrate that produces a visible or fluorescent precipitate uponenzyme action (for example, the action of horseradish peroxidase upondiaminobenzidine, or enzyme action on a labeled tyramide), visible- orfluorescent-labeled microparticles, a metal such as colloidal gold, or asilver-containing reagent, or a signal that is released by the action oflight upon the reagent (e.g. a caged fluorophore that is activated byphotolysis, or the action of light upon diaminobenzidine), or aradioactive signal. The detectable signal of the additional reagent isdetected simultaneously or sequentially with the optical signal of theconjugates of the present invention.

In one embodiment of the invention, the additional detection reagent isselected to exhibit overlapping spectral characteristics, such thatenergy transfer occurs between the reagent-target conjugate and thedetection reagent, resulting in labeled targets that exhibit an extendedStokes shift. Alternatively, the additional detection reagentcolocalizes with the reagent-target conjugate such that the labeling ofsome or all targets exhibit quenching.

One class of useful additional detection reagents are fluorescentnucleic acid stains. A variety of appropriate nucleic acid stains areknown in the art, including but not limited to, thiazole orange,ethidium homodimer, ethidium bromide, propidium iodide, Hoechst 33258,and DAPI. Additional useful nucleic acid stains are described in theinternational applications WO 93/06482, DIMERS OF UNSYMMETRICAL CYANINEDYES (published Apr. 1, 1993) or WO 94/24213, CYCLIC SUBSTITUTEDUNSYMMETRICAL CYANINE DYES (published Oct. 27, 1994); U.S. Pat. No.5,321,130 to Yue et al., 1994; or U.S. Pat. No. 5,410,030 to Yue et al.,1995 (all incorporated by reference). The use of an appropriate nucleicacid stain in conjunction with the reagents of the present inventionpermits simultaneous or sequential observation of the desired target andnucleic acids such as DNA and RNA.

Another class of useful additional detection reagents are fluorescentprotein stains. A variety of appropriate protein stains are known in theart, including but not limited to, those described in the internationalapplication WO 00/25139, LUMINESCENT PROTEIN STAINS AND THEIR METHOD OFUSE (published May 4, 2000), or U.S. Pat. No. 5,616,502 to Haugland etal., 1997; incorporated by reference. Selected preferred fluorescentprotein stains are sold under the trademarks SYPRO RED, SYPRO ORANGE,SYPRO ROSE, SYPRO ROSE PLUS, and SYPRO RUBY by Molecular Probes, Inc.(Eugene, OR). The use of an appropriate protein stain in conjunctionwith the reagents of the present invention permits simultaneous orsequential observation of the desired target and total protein presentin the sample.

An additional class of useful additional detection reagents arefluorescent-labeled antibodies, lectins or avidins, in particular thosethat are labeled with an OREGON GREEN dye or ALEXA FLUOR dye,commercially available from MOLECULAR PROBES, INC., Eugene, Oreg.

In one embodiment, the additional reagent comprises a member of aspecific binding pair having a detectable label. Representative specificbinding pairs are shown in Table 1.

TABLE 1 Representative specific binding pairs enzyme enzyme substrateantigen antibody biotin avidin (or streptavidin) IgG* protein A orprotein G carbohydrate lectin nucleic acid aptamer protein *IgG is animmunoglobulin

The additional detection reagent is used in conjunction with enzymeconjugates to localize the detectable response of the additionalreagent. Enzyme-mediated techniques take advantage of the attractionbetween specific binding pairs to detect a variety of analytes. Ingeneral, an enzyme-mediated technique uses an enzyme attached to onemember of a specific binding pair or series of specific binding pairs asa reagent to detect the complementary member of the pair or series ofpairs. In the simplest case, only the members of one specific bindingpair are used. One member of the specific binding pair is the analyte,i.e. the substance of analytical interest. An enzyme is attached to theother (complementary) member of the pair, forming a complementaryconjugate. Alternatively, multiple specific binding pairs may besequentially linked to the analyte, the complementary conjugate, or toboth, resulting in a series of specific binding pairs interposed betweenthe analyte and the detectable enzyme of the complementary conjugateincorporated in the specific binding complex.

Additional classes of preferred detection reagents include labeledantibodies, ion indicators (including Na⁺ indicators, Ca²⁺ indicators,or pH indicators), organelle stains, cell viability indicators, orchemically reactive dyes.

Illumination and Observation

At any time after or during staining, the sample is illuminated with awavelength of light capable of exciting the reagent to produce adetectable optical response, and observed with a means for detecting theoptical response. Equipment that is useful for illuminating the dyecompounds of the invention includes, but is not limited to, hand-heldultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laserdiodes. These illumination sources are optionally integrated into laserscanners, fluorescence microplate readers, standard or minifluorometers,or chromatographic detectors.

A detectable optical response means a change in, or occurrence of, anoptical signal that is detectable either by observation orinstrumentally. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

The presence of the optical response is optionally used to identify thepresence of the analyte in the test sample. Alternatively, thedetectable optical response is quantified and used to measure theconcentration of the target in the sample mixture. Quantification istypically performed by comparison of the optical response to a preparedstandard or to a calibration curve. Typically, the measured opticalresponse is compared with that obtained from a standard dilution of aknown concentration of the target in an electrophoretic gel, or on amembrane. Generally a standard curve must be prepared whenever anaccurate measurement is desired. Alternatively, the standard curve isgenerated by comparison with a reference dye or dyed particle that hasbeen standardized versus the reagent-target conjugate desired.

In one aspect of the invention, stained electrophoretic gels are used toanalyze the composition of complex sample mixtures and additionally todetermine the relative amount of a particular target in such mixtures.

Comparison/Differentiation Assays

The method of the invention further comprises assays wherein twosamples, or two aliquots of the same sample, are each treated with areagent of the invention and the optical response of each sample and/oraliquot is then compared.

In one aspect of the method, each aliquot is from the same originalsample, but one aliquot is treated with a reagent or exposed to acondition, while the other is used as a control. In another aspect ofthe method, each sample has a different composition, and the samples arecompared after both are treated with a reagent of the invention. In afurther aspect of the invention, the sample is polydisperse, as in thecase of cells and tissues, and the targets are spatially resolved. Thesamples are optionally exposed to an oxidizing condition, treated withan acid or an enzyme prior to adding a staining solution. In addition,each sample is optionally subjected to a separation step before or aftertreatment with a staining solution.

Selected embodiments of this method include:

-   Comparing the electrophoretic mobility of the components of two    treated samples to highlight differences in the samples.-   Distinguishing between Gram-positive and Gram-negative bacteria by    detecting the lipooligosaccharides expressed on the surface of    mucosal Gram-negative bacteria (see Example 26).-   Comparing a sample treated with a glycosidase enzyme with an    appropriate negative control.-   Comparing proteins treated with galactose oxidase with an    appropriate negative control.-   Comparing cells treated with functionalized glycoconjugates with an    appropriate negative control.-   Differentiating cells based on DNA content (after treatment by the    Feulgen reaction).-   Differentiating cell surface proteins that have been subjected to an    oxidizing condition from internal proteins that have not been    oxidized.-   Differentiating normal and tumor cells.-   Differentiating glycosylated albumins from nonglycosylated albumins-   differentiating types of blood cells by differences in carbohydrate    content, optionally using flow cytometry    Kits

Due to the simplicity of use of the instant reagents, they areparticularly useful in the formulation of a kit for the labeling ofselected targets, comprising one or more reagents (preferably in a stocksolution), instructions for the use of the reagent to label or detect adesired target, and optionally comprising target standards and othercomponents (such as inorganic salts, buffers, or wash solutions). In oneembodiment, the kit of the invention comprises a stock solution of areagent of the invention and one or more additional kit components.

The additional kit components may be selected from, without limitation,acids, buffering agents, inorganic salts, polar solvents, and positiveand negative controls. The additional kit components are present as purecompositions, or as aqueous solutions that incorporate one or moreadditional kit components. Any or all of the kit components optionallyfurther comprise buffers. Where the additional kit component is aninorganic salt, it is typically a sodium or magnesium salt.

In one aspect of the invention, the kit contains an oxidizing agent(preferably a periodate salt), a stock solution of a reagent of theinvention in an organic solvent, a buffer solution that additionallycomprises an inorganic salt, molecular weight standards, and optionallyan additional detection reagent that is a fluorescent total proteinstain. Preferably, the compound in the staining solution is Formula XXX.In another aspect of the invention, the staining solution in the kitcontains Compound 5, 20, 21, or 23; preferably 5, 20 or 23.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

EXAMPLE 1 Preparation of Compound 1

Anthranilamide (0.2 mol) and 2-nitrobenzaldehyde (0.22 mol) aredissolved in anhydrous ethanol (EtOH, 1 L). To the EtOH solution isadded p-toluenesulfonic acid monohydrate (TsOH, 0.01 mol). The reactionmixture is stirred at room temperature for 2 h, and then refluxed untilanthranilamide is completely consumed. The reaction mixture is cooled toroom temperature, and directly used for the next step reaction withoutfurther purification.

EXAMPLE 2 Preparation of Compound 2

To the solution of crude Compound 1 prepared in Example 1 (0.2 mol) isslowly added 2,3-dichloro-5,6-dicyano-1,4-quinone (DDQ, 0.21 mol) atroom temperature. The reaction mixture is stirred at room temperatureuntil Compound 1 is completely consumed. The reaction mixture isconcentrated in vacuo, and filtered to collect the resultingprecipitate. The crude solid is washed with cold 1:1 EtOH/benzene untilthe residual DDQ and 2,3-dichloro-5,6-dicyano-1,4-dihydroquinone arecompletely removed. The crude material is further recrystallized (fromEtOH/ethyl ether) to give the desired product (51 g).

EXAMPLE 3 Preparation of Compound 3

To a solution of Compound 2 (0.1 mol) in tetrahydrofuran (500 mL) isslowly added tin (II) chloride (0.5 mol) at room temperature. Thereaction mixture is heated at 50-60° C. until compound 2 is completelyconsumed. The solution is carefully poured onto crushed ice/2 M HCl, andfiltered to collect the resulting solid. The crude solid is suspended inwater, and neutralized with NaHCO₃ to pH 8.0-9.0. The resultingsuspension is filtered to collect the solid. The crude material iswashed with water, dried and recrystallized to give the desired product(24 g).

EXAMPLE 4 Preparation of Compound 4

To a solution of Compound 3 (10 mmol) in N,N-dimethylformamide (DMF, 50mL) is slowly added 4-fluorosulfonylbenzoyl chloride (11 mmol) at roomtemperature. The reaction mixture is stirred at room temperature untilCompound 3 is completely consumed. The solution is then carefully pouredonto crushed ice/water, and filtered to collect the resulting solid. Thecrude solid is redissolved in DMF, and precipitated with ethyl ether.The solubilization/precipitation process is repeated twice to give thedesired product (3.8 g).

EXAMPLE 5 Preparation of Compound 5

To a methanol solution of anhydrous hydrazine (50 mmol, 5 mL) is slowlyadded a DMF solution of Compound 4 (5 mmol, 5 mL). The reaction mixtureis stirred at room temperature until Compound 4 is completely consumed.The solution is concentrated in vacuo, poured into water, and filteredto collect the resulting solid. The crude solid is redissolved in DMF,and precipitated with water. The solubilization/precipitation process isrepeated until the residual hydrazine is completely removed (TLCdetection by ninhydrin spraying). The crude material is recrystallizedfrom EtOH to give the desired product which has yellow/greenfluorescence (1.8 g).

EXAMPLE 6 Preparation of Compound 6

To a THF suspension of Compound 3 (0.5 mmol, 5 mL) is slowly addedthiophosgene (5 mmol, 5 mL). The reaction mixture is heated at refluxuntil Compound 3 is completely consumed. The resulting solution isevaporated in vacuo, and the residue is washed with ether. The crudesolid is directly used for the next step reaction without furtherpurification.

EXAMPLE 7 Preparation of Compound 7

To a DMF solution of anhydrous hydrazine (5 mmol, 0.5 mL) is slowlyadded a DMF solution of Compound 6 (0.5 mmol, 0.5 mL). The reactionmixture is stirred at room temperature until Compound 6 is completelyconsumed. The solution is poured into water, and filtered to collect theresulting solid. The crude solid is redissolved in DMF, and precipitatedwith water. The solubilization/precipitation process is repeated untilthe residual hydrazine is completely removed. The crude material isrecrystallized from EtOH to give the desired product, which has bluefluorescence (1.8 g).

EXAMPLE 8 Preparation of Compound 8

Compound 8 is prepared analogously to Compound 1 using5-nitroanthranilamide and salicyaldehyde.

EXAMPLE 9 Preparation of Compound 9

Compound 9 is prepared by oxidation of Compound 8 as in the procedure ofCompound 2.

EXAMPLE 10 Preparation of Compound 10

Compound 10 is prepared by reduction of Compound 8 as in the procedureof Compound 3. Compound 10 exhibits green fluorescence.

EXAMPLE 11 Preparation of Compound 11

To a cold suspension of Compound 10 (0.1 mmol) in concentratedhydrochloric acid (10 mL) is slowly added 95% NaNO₂ (0.11) whilemaintaining the reaction temperature at 0-5° C. The reaction mixture isstirred at room temperature until Compound 10 is completely consumed.The reaction mixture is checked with starch-iodide paper for excessnitrous acid, which is then destroyed by adding small quantities ofurea. The resulting solution is directly used in the next reaction.

EXAMPLE 12 Preparation of Compound 12

To the solution of crude Compound 11 prepared above (0.1 mol) is slowlyadded 5 M aqueous tin (II) chloride (0.3 mol, in concentrated HCl) atroom temperature. The reaction mixture is stirred at room temperatureuntil compound 11 is completely consumed. The reaction mixture isdiluted with water, and filtered to yield the resulting precipitate,which is repeatedly washed with water to remove the residual tinchloride. The resulting solid is suspended in water, and neutralizedwith NaHCO3 (pH 7.0-7.5). The mixture is filtered to collect theprecipitate that is further washed with water. The solid is dried, andrecrystallized from EtOH to give the desired product, which has greenfluorescence (67 mg).

EXAMPLE 13 Preparation of Compound 13

Anthranilic acid (10 mmol) and 2-hydroxybenzaldehyde (12 mmol) aredissolved in polyphosphoric acid (10 mL). The reaction mixture is heatedat 60-70° C. for 4-6 h. The solution is then cooled to room temperature,and poured into ice/water. The resulting precipitate is collected byfiltration, and washed with water. The crude material is furtherpurified on a silica gel column eluting with 5:1 chloroform/ethylacetate to give the desired product (1.2 g).

EXAMPLE 14 Preparation of Compound 14

To a methanol solution of anhydrous hydrazine (5 mmol, 5 mL) is slowlyadded a methanol solution of Compound 13 (1 mmol, 5 mL). The reactionmixture is then heated to 50-60° C. until Compound 13 is completelyconsumed. The solution is then concentrated in vacuo, poured into water,and filtered to collect the resulting solid. The crude solid isdissolved in DMF, and precipitated with water. Thesolubilization/precipitation process is repeated until the residualhydrazine is completely removed. The crude material is recrystallizedfrom ethyl acetate to give the desired product, which has bluefluorescence (1.8 g).

EXAMPLE 15 Preparation of Compound 15

Compound 15 is prepared as in the procedure for Compound 4 usingCompound 3 and hexafluoroglutaric anhydride.

EXAMPLE 16 Preparation of Compound 16

To a methanol solution of Compound 15 (1 mmol, 25 mL MeOH) is added 0.1mL concentrated sulfuric acid. The reaction mixture is heated at 50-60°C. until Compound 15 is completely consumed. The resulting solution isconcentrated in vacuo, poured into water, and filtered to collect theresulting solid. The crude solid is washed with water until pH 6.0-7.0,and purified on a silica gel column, eluting with a gradient mixture ofchloroform/methanol.

EXAMPLE 17 Preparation of Compound 17

To a methanol solution of anhydrous hydrazine (5 mmol, 5 mL) is slowlyadded a methanol solution of Compound 16 (1 mmol, 5 mL). The reactionmixture is stirred at room temperature until Compound 16 is completelyconsumed. The resulting solution is concentrated in vacuo, poured intowater, and filtered to collect the resulting solid. The crude solid isredissolved in methanol, and precipitated with water. Thesolubilization/precipitation process is repeated until the residualhydrazine is completely removed. The crude material is recrystallizedfrom ethyl acetate to give the desired product, which exhibits greenfluorescence (213 mg).

EXAMPLE 18 Preparation of Compounds 20-34

Compounds 20-34 are prepared according to the procedures indicated inTable 2.

TABLE 2 Prepared as in Compound Example: Fluorescence color

4 yellow

4 yellow/green

4 green

4 yellow

4 yellow/green

4 yellow/green

12  green/blue

4 green

4 blue

4 yellow/green

12  green/blue

12  blue

12  green

12  yellow

12  yellow/orange

4 yellow/green

EXAMPLE 19 Detection of Glycoproteins in SDS Polyacrylamide Gels

Proteins of interest were separated by SDS-polyacrylamide gelelectrophoresis utilizing a 4% T, 2.6% C stacking gel, pH 6.8 and 15% T,2.6% C separating gel, pH 8.8, according to standard procedures. % T isthe total monomer concentration (acrylamide +crosslinker) expressed ingrams per 100 mL and % C is the percentage crosslinker (e.g.N,N′-methylene-bis-acrylamide, N,N′-diacryloylpiperazine or othersuitable agent). After electrophoresis, gels were incubated in 50%methanol for 30 minutes, in two changes of 3% acetic acid for 5 minutes,and then in 1% periodic acid prepared in 3% acetic acid for 30 minutes.Alternative oxidizing agents might include chromic acid, permanganate,lead tetraacetate, sodium bismuthate, manganese acetate or phenyliodoacetate. Gels were then washed in 3% acetic acid for 4×10 minutes,and incubated for 30-120 minutes in a solution of 5 μM Compound 5, 3%acetic acid, 2% dimethylformamide, and 0.25 M magnesium chloride. Thesalt may be omitted, but staining intensity is decreased. Other saltsmay be used instead of magnesium chloride, including sodium chloride andmagnesium sulfate. After labeling with the reagent, gels were rinsed in3% acetic acid twice for 5 minutes each. Glycoproteins may be viewedusing a 300 nm UV transilluminator. Proteins appeared as greenluminescent bands on a clear background (Fluorescenceexcitation/emission spectra for Compound 5-labeled α1-acid glycoproteinare given in FIG. 1).

EXAMPLE 20 Detection of Glycoproteins in Isoelectric Focusing Gels

Isoelectric focusing (IEF) can be performed utilizing a variety ofpre-cast and laboratory prepared gels that employ different chemistriesto generate a pH gradient. In this instance, Ampholine PAG plates wererun horizontally for 1500 volt-hours using a Multiphor IIelectrophoresis unit (Amersham-Pharmacia Biotech, Uppsala, Sweden) perthe manufacturer's instructions. In another alternative, denaturing, 1mm IEF slab gels were cast utilizing a 4% T, 2.6% C polyacrylamide gelmatrix, containing 9 M urea, 2% Triton X-100, and 2% carrier ampholytes.Electrophoresis was performed on a Multiphor II electrophoresis unit for1500 volt-hours using 10 mM phosphoric acid and 100 mM sodium hydroxideas anode and cathode buffer, respectively. Luminescent staining ofglycoproteins in gels was performed by immersing the gel in 50% methanolfor 30 minutes, in two changes of 3% acetic acid for 5 minutes, and thenin 1% periodic acid in 3% acetic acid for 30 minutes. Gels were thenwashed in 3% acetic acid for 4×10 minutes, then incubated for 30-120minutes in a solution of 5 μM Compound 23, 3% acetic acid, 2%dimethylformamide, and 0.25 M magnesium chloride. Gels were then rinsedin two changes of water for one hour each and viewed by illuminationwith a 300 nm UV light source. Glycoproteins appeared as greenluminescent bands on a clear background.

EXAMPLE 21 Detection of Glycoproteins in Two-Dimensional Gels

A mouse 3T3 fibroblast cell lysate protein mixture was solubilized in 8M urea, 2% Triton X-100, 2% carrier ampholytes, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate, 12.5 mM Tris, pH 8.0. Approximately 50 μgof protein was applied to 1 mm diameter, 20 cm long isoelectric focusinggels consisting of a 4% T, 2.6% C polyacrylamide gel matrix, containing9 M urea, 2% Triton X-100, and 2% carrier ampholytes. Gels were runvertically for 18,000 volt-hours using 10 mM phosphoric acid and 100 mMsodium hydroxide as anode and cathode buffer, respectively. Isoelectricfocusing gels were incubated in 0.3 M Tris base, 0.075 M Tris-HCl, 3%SDS, 0.01% bromophenol blue for two minutes. Isoelectric focusing gelswere then laid on top of 1 mm thick, 20 cm×20 cm, 12.5% T, 2.6% Cpolyacrylamide gels containing 375 mM Tris-base, pH 8.8 andSDS-polyacrylamide gel electrophoresis was performed according tostandard procedures except that the cathode electrode buffer was 50 mMTris, 384 mM glycine, 4% sodium dodecyl sulfate, pH 8.8 while the anodeelectrode buffer was 25 mM Tris, 192 mM glycine, 2% sodium dodecylsulfate, pH 8.8. After the second dimension electrophoresis, gels wereincubated in 50% methanol for 30 minutes, in two changes of 3% aceticacid for 5 minutes, and then in 1% periodic acid in 3% acetic acid for30 minutes. Gels were then washed in 3% acetic acid for 4×10 minutes,then incubated for 30-120 minutes in a solution of 5 μM Compound 5, 3%acetic acid, 2% dimethylformamide, and 0.25 M magnesium chloride. Gelswere rinsed in dd H₂O for 10-15 minutes and viewed using a 300 nm UVtransilluminator. Glycoproteins appeared as green luminescent spots on aclear background. The majority of glycoproteins were observed to have anisoelectric point that was less than 5.5 in this sample.

EXAMPLE 22

Serial dichromatic detection of glycosylated and nonglycosylatedproteins in two-dimensional gels.

Two-dimensional gel electrophoresis was performed as described inExample 21. Glycoprotein detection was performed in the same manner aswell. The signal from the green fluorescent glycoproteins was collectedwith a standard CCD camera-based imaging system using a 520 nm bandpassfilter. After detection of the glycoproteins, the gel was stained withSYPRO RUBY protein gel stain (Molecular Probes, Eugene, Oreg.) byincubating the gel for 3-20 hours in the stain, and then incubating thegel in 7% acetic acid, 10% methanol for 30 minutes. The orange signalfrom the glycosylated and nonglycosylated proteins was collected with astandard CCD camera-based imaging system with a 600 nm bandpass filteror a 610 nm longpass filter.

EXAMPLE 22a Dichromatic Detection of Glycosylated and NonglycosylatedProteins in Two-Dimensional Gels

Alternatively, the two-dimensional gels may be stained with SYPRO RUBYprotein gel stain first, followed by detection of glycoproteins usingCompound 5. Both reagents were excited by 300 nm UV illumination buttheir emission maxima differ by 80 nm. Upon illumination using astandard UV transilluminator glycoproteins appeared green whilenonglycosylated proteins appeared orange. Using a standard CCDcamera-based imaging system, the two signals may be imaged separatelyusing a 520 nm band pass filter to collect signal from the Compound 5and a 640 nm band pass filter to collect the signal from SYPRO RUBYprotein gel stain.

EXAMPLE 23 Detection of Glycoproteins Electroblotted to PVDF orNitrocellulose Membranes

Proteins of interest were separated by SDS-polyacrylamide gelelectrophoresis and transferred to PVDF or nitrocellulose membrane usingstandard procedures. Following dot- or slot-blotting, membranes wereallowed to air dry to minimize loss of protein during subsequentstaining steps. The membrane was subsequently immersed in 50% methanol(for PVDF membrane) or 10% methanol (for nitrocellulose membrane) for 30minutes, in two changes of 3% acetic acid for 5 minutes, and then in 1%periodic acid in 3% acetic acid for 30 minutes. The blots were thenwashed in three or four changes of 3% acetic acid for 5-10 minutes.Blots were then incubated for 30-120 minutes in a solution of 5 μMCompound 5, 3% acetic acid, 2% dimethylformamide, and 0.25 M magnesiumchloride. The membrane was incubated for 5 minutes each in four changesof dd-H₂O. The membrane was allowed to air dry and was subsequentlyviewed using a reflective or transmissive 300 nm UV light source.Glycoproteins appeared as bright green luminescent bands on a faint blueor green background. The signal from the green fluorescent glycoproteinswas collected with a standard CCD camera-based imaging system using a520 nm bandpass filter. Serial dichromatic detection of glycosylated andnonglycosylated proteins on PVDF membranes was accomplished by floatingthe membrane face down on a solution of 10% methanol, 7% acetic acid for10 minutes followed by face staining with SYPRO RUBY blot stain for 15minutes. The membrane was washed face down on water, 3 changes in 5minutes. The membrane was allowed to air dry. The fluorescent signalfrom total proteins was detected using a standard CCD camera-basedimaging system with a 520 nm longpass filter. Alternatively dichromaticdetection of nonglycosylated proteins and glycoproteins was accomplishedwith the orange fluorescent signal due to SYPRO Ruby stain collectedusing a 640 nm longpass filter, and the green signal due toglycoproteins stained with Compound 5 was collected using a 520 nmbandpass filter. Alternatively, a PVDF membrane containingelectroblotted proteins was stained with SYPRO RUBY protein blot stainfollowed by staining with Compound 5, as above, and serial dichromaticstaining accomplished as above.

EXAMPLE 24 Mobility-Shift Gel Analysis Using Deglycosylating Enzymes andDetection with either SYPRO RUBY Protein Gel stain for Visualization ofTotal Proteins or a Reagent of the Invention for Detection ofGlycosylated Proteins

In the assay, glycoproteins were incubated with glycosidase enzymes andcompared with undigested proteins using SDS-polyacrylamide gelelectrophoresis. Deglycosylation was performed by standard methods usingcommercially available kits such as the Prozyme Glycopro Deglycosylationkit (Prozyme Inc, San Leandro, Calif.). Typically, these kits containPNGase F to remove N-linked glycans, Endo-O-Glycosidase to removeO-linked glycans and Sialidase A to remove sialic acid residues.Control, undigested proteins were loaded into lanes of a standard 13%SDS-polyacrylamide gel as well as the corresponding proteins afterincubation with glucosidases. Suitable test proteins include α1-acidglycoprotein, horseradish peroxidase, bovine fetuin; ovalbumin and PHA-Mlectin. SDS-polyacrylamide gel electrophoressis was performed bystandard procedures. Serial dichromatic staining was performed asdescribed in Example 22. The signal from the green fluorescentgylcoproteins was collected with a standard CCD camera-based imagingsystem using a 520 nm bandpass filter. After detection of theglycoproteins, the gel was stained with SYPRO RUBY protein gel stain(Molecular Probes, Eugene, Oreg.) by incubating the gel for 3-20 hoursin the stain, and then incubating the gel in 7% acetic acid, 10%methanol for 30 minutes. The orange signal from the glycosylated andnonglycosylated proteins was collected with a standard CCD camera-basedimaging system with a 600 nm bandpass filter or a 610 nm longpassfilter. Without exposure to glycosidases, the glycoproteins α1-acidglycoprotein, horseradish peroxidase, fetuin, ovalbumin and PHA-M lectinwere detected with both stains. After glycosidase treatment, α1-acidglycoprotin, fetuin, ovalbumin and PHA-M showed a marked mobility shiftas detected by SYPRO RUBY protein gel staining and loss of staining bythe reagent of the invention, indicating that the carbohydrate groupswere cleaved off. However, the horseradish peroxidase did not show asubstantial mobility shift, although staining with Compound 5 shows thatit contained a significant amount of carbohydrate. This proteincontained an α-(1,3)-fucosylated asparagine N-acetylglucosamine-linkagethat was resistant to cleavage by the glycosidases used in this study.Thus, the use of the Compound 5 identified glycoproteins not susceptibleto glycosidases used in the mobility-shift assay, providing importantinformation about the glycoprotein's carbohydrate structure.

EXAMPLE 25 Detection of Glycoprotein in Filtration Plates

Prior to protein application the hydrophobic membranes in individualwells of a 96-well Millipore MultiScreen filtration plate are wettedwith methanol and then rinsed with 7% acetic acid using a vacuummanifold per manufacturer's instructions (Millipore Corporation,Bedford, Mass.). 0.2 to 1000 ng/mm² of ovalbumin is applied toindividual wells without application of a vacuum. The plate is incubatedfor 30-60 minutes before protein is removed by application of a vacuum.The filtration plate is then allowed to air dry and wells are incubatedin 50% methanol for 30 minutes, in two changes of 3% acetic acid for 5minutes, and then in 1% periodic acid in 3% acetic acid for 30 minutes.Wells are then rinsed in 3% acetic acid and incubated for 30 minutes ina solution of 5 μM Compound 5, 3% acetic acid, 2% dimethylformamide, and0.25 M magnesium chloride. The dye solution is removed from the wells bypipetting and 200 μL of 3% acetic acid is applied and removed bypipetting 3-4 times to remove any unbound dye. The filtration plate issubsequently read using a Perkin-Elmer HTS 7000 microplate reader orsimilar device. An excitation filter of 300 nm and an emission filter of530 nm is selected. Measurements are made through the top face of theplate. Instrument software provides digital values corresponding to theluminescence intensity of the signal from the dye in each well.

EXAMPLE 26 Detection of Lipopolysaccharides in SDS Polyacrylamide Gels

A serial dilution of Escherichia coli serotype 055:B5 lipopolysaccharideranging from 1 to 4000 nanograms was separated by SDS gelelectrophoresis using a 15% T, 2.6% C polyacrylamide gel. The gel wassubsequently immersed in 50% methanol for 30 minutes, in two changes of3% acetic acid for 5 minutes, and then in 1% periodic acid in 3% aceticacid for 30 minutes. Gels were rinsed 4×5 minutes in 3% acetic acid,then incubated for 30 minutes in a solution of 5 μM Compound 5, 3%acetic acid, 2% dimethylformamide, and 0.25 M magnesium chloride.Afterwards, the gel was rinsed in 3% acetic acid for 5 minutes andplaced on a 300 nm UV transilluminator. The separatedlipopolysaccharides appeared as bright green luminescent bands withlittle or no background signal. The capability of Compound 5 to detectbacterial lipopolysaccharides was compared with that of an alkalinesilver diamine-based stain (Silver Stain Plus, Bio-Rad Laboratories,Hercules, Calif.). The alkaline silver diamine stain was selectedbecause it is superior to an acidic silver nitrate stain, zinc-imidazolestain, SYPRO ORANGE stain and SYPRO RUBY protein gel stain in terms ofits ability to detect lipopolysaccharides. Detection of as little as 2-8ng of total applied lipopolysaccharide after SDS-polyacrylamide gelelectrophoresis was possible using Compound 5 while only 250-1000 ng canbe detected using the silver staining method. Using Compound 5, thelinear dynamic range for detection of lipopolysaccharide extended from2-4000 ng of applied material (r²=0.9901). Similar results were obtainedwhen lipopolysaccharides from Pseudomonas aeruginosa, serotype 10 orEscherichia coli EH100 (RA mutant) are evaluated. It should be notedthat the lipolysaccharides typically separate into roughly 20 bands, andthat the detection sensitivity of the individual components wassubstantially below a single nanogram using Compound 5.Lipooligosaccharides, the major glycolipids expressed on mucosalGram-negative bacteria may be detected by similar procedures, and mayprovide a means to distinguish Gram positive and Gram negative bacteria.

EXAMPLE 27 Detection of Glycogen Slot-Blotted onto PVDF Membranes

A serial dilution of glycogen was prepared in deionized water or othersuitable solution such as 7% acetic acid or 20 mM Tris HCl, pH 6.8, 500mM NaCl. For dot-blotting, 1-5 μL volumes of the glycopolymer areapplied to a 0.4 μm pore size wetted PVDF membrane using a pipetter.Slot-blotting was performed using a Bio-Dot SF vacuum apparatus (Bio-RadLaboratories, Hercules, Calif.). For slot-blotting, membranes wererehydrated with methanol, then 100 μL/well dd-H₂O, samples were appliedto the membranes (200 μL/well), wells were rinsed twice with 600 μL of7% acetic acid/10% methanol and twice with 600 μL of dd-H₂O. Followingdot- or slot-blotting, membranes were allowed to air dry to minimizeloss of polymer during subsequent staining steps. The membrane wassubsequently immersed in 50% methanol for 30 minutes, in two changes of3% acetic acid for 5 minutes, and then in 1% periodic acid in 3% aceticacid for 30 minutes. Blots were rinsed 4×5 minutes in 3% acetic acid,and then incubated for 30-120 minutes in a solution of 5 μM Compound 5,3% acetic acid, 2% dimethylformamide, and 0.25 M magnesium chloride. Themembrane was incubated for 5 minutes each in four changes of dd-H₂O. Themembrane was allowed to air dry and was subsequently viewed using areflective or transmissive 300 nm UV light source. Spotted glycogenappeared as green luminescent bands on a faint fluoroescent bluebackground. The limits of detection is typically 4-8 ng of glycogen andthe linear dynamic range of detection extends from 16 to 250 ng ofapplied material (r²=0.958). Other glycopolymers such as starch, chitin,cellulose and pectin should be detectable using similar methods.Parallel experiments performed with chondroitin-4-sulfate reveal thatthe detection sensitivity for this glycosaminoglycan is quite poor, withlimits of detection in the vicinity of 16 μg of applied material. Thisis not unexpected as glycosaminoglycans such as chondroitin sulfate,hyaluronic acid, dermatan sulfate, alginates, fucoidan, carrageenans andkeratan sulfate are known to stain poorly by conventional periodic acidSchiffs procedures.

EXAMPLE 28 Pre-Derivatization of Glycoproteins Followed bySDS-Polyacrylamide Gel electrophoresis

A glycoprotein such as horseradish peroxidase is diluted to 5 mg/mL in100 mM sodium acetate buffer, pH 5.0 containing 10 mM in sodiumperiodate and then the sample is incubated in the dark for 2 hours. Thesample is then diluted 2 fold into a solution of 10 μM Compound 5, 6%acetic acid, 4% dimethylformamide, and 0.5 M magnesium chloride andincubated for 30 minutes to 2 hours. Afterwards, the protein sample isadded to a sufficient amount of ice-cold acetone to yield an 80% acetonesolution. After incubating on ice for 10 minutes, the sample iscentrifuged at 14,000×G using a tabletop microcentrifuge, the supernateis discarded and the pellet is resuspended in standard SDS samplebuffer. The sample is heated to 100° C., cooled to room temperature andthen applied to a 13% T, 2.6% C SDS-polyacrylamide gel. Electrophoresisis performed by standard methods and the gel is then placed upon a 300nm UV transilluminator. The glycoprotein appears as a green fluorescentband in the gel.

EXAMPLE 29 Specific Detection of DNA after Polyacrylamide GelElectrophoresis

Two-fold serial dilutions of Hae III-cleaved φX174 RF DNA starting with200 ng as well as 2-fold serial dilutions of ribosomal RNA starting at500 ng were prepared in native loading buffer (5% glycerol),heat-denatured at 95° C., quick-cooled on ice, loaded onto 0.75 mm thick5% T, 5% C polyacrylamide gels and separated by electrophoresis for 3-4hours in 89 mM Tris borate, 2 mM EDTA, pH 8.3 (TBE buffer) at 60 voltsby standard procedures. After electrophoresis, gels were incubated for30 minutes in 50% methanol, and then incubated in 5 M HCl at roomtemperature for 20 minutes. Gels were incubated in 3% acetic acid for 5minutes and then incubated in 5 μM Compound 23, 3% acetic acid, 2%dimethylformamide, and 0.25 M magnesium chloride for 30-120 minutes.Gels were then rinsed in 3% acetic acid and placed onto a 300 nm UVtransilluminator. 1 ng of DNA was readily detected by this method asfluorescent green bands. In comparison, ribosomal RNA (500 ng) was notlabeled by this method. The cited labeling procedure was based upon thehistological Feulgen reaction, where hydrolysis by HCl frees thealdehyde groups of deoxyribose, and may also be used to selectivelydetect DNA in cells. An intensity profile of electrophoreticallyseparated DNA molecular weight standards using Compound 5 is given inFIG. 7.

EXAMPLE 30 Detection of both DNA and RNA after Polyacrylamide GelElectrophoresis

Dilution series of DNA and RNA are prepared and subjected toelectrophoresis as described in Example 29. Gels are then incubated in100 mM sodium acetate buffer, pH 4.5 containing 1 M sodium bisulfite, 10μM Compound 5. The gels are incubated at 37° C. for 24 hours. Cytosineresidues are converted to 6-sulfo-cytosine residues and Compound 5 isthen incorporated by a transamination reaction. Gels are incubated in 3%acetic acid for 5 minutes and then placed on a 300 nm UVtransilluminator. Both DNA and RNA appear as fluorescent green bands ona pale blue fluorescent background.

EXAMPLE 31 Immunodetection of Tubulin Using Oxidized HRP-ConjugatedSecondary antibody

Approximately 1-2000 ng of bovine brain tubulin prepared in a constantamount of broad range molecular weight standards (300 ng/protein band)was applied per gel lane and separated by SDS-polyacrylamide gelelectrophoresis utilizing a 4.25% T, 2.6% C stacking gel, pH 6.8 and 13%T, 2.6% C separating gel, pH 8.8 by standard methods. Gels weretransferred to PVDF membranes by standard methods. Blots weresubsequently incubated three times for 10 minutes each in modifiedTris-buffered saline equilibration buffer (MTBS; 50 mM Tris base, 150 mMNaCl, pH 7.5) and then for 60 minutes in MTBS containing 0.2% Tween-20and 0.5% poly(vinylalcohol) (PVA; 30,000-70,000 Da, Sigma Chemical).Monoclonal anti-tubulin antibody (Molecular Probes, Eugene, Oreg.) wasdiluted 1:500 in MTBS/Tween-20/PVA and blots were incubated in thesolution overnight. Blots were then rinsed in MTBS/Tween-20/PVA 4 timesfor 10 minutes each. Blots were then incubated for 60 minutes in1:10,000 dilution of oxidized horseradish peroxidase-conjugatedsecondary antibody prepared in MTBS/Tween-20/PVA. Oxidation of theconjugate was accomplished by incubating 5 mg/mL of the proteinconjugate in 100 mM sodium acetate buffer, pH 4.5 containing 10 mMsodium periodate for 2 hours in the dark. The reaction was terminated bythe dilution step prior to application to the membrane. After incubatingin the secondary antibody, blots were incubated in four changes ofMTBS/Tween-20/PVA for 5 minutes each and then in 5 μM Compound 5, 3%acetic acid, 2% dimethylformamide, and 0.25 M magnesium chloride. Themembrane was rinsed in 3% acetic acid for 10 minutes and fluorescentsignal was visualized using a Lumi-Imager CCD-based computerized imageanalysis system (Roche Biochemicals, Indianapolis, Ind.). Tubulinappeared as a white band against a black background on the computermonitor.

EXAMPLE 32 Converting Compound 5 into a Red-Emitting Stain

An SDS-polyacrylamide gel containing a mixture of glycosylated andunglycosylated protein molecular weight standards was treated withCompound 5 as described in Example 19. The gel was incubated in 3%acetic acid for 10 minutes and then incubated in a 1:5000 dilution ofSYPRO RED protein gel stain (Molecular Probes, Eugene, Oreg.) preparedin 3% acetic acid, 0.001% SDS. The gel was incubated in the stainingsolution for 90 minutes and then briefly rinsed in 3% acetic acid. Usinga UV transilluminator, glycoproteins appeared as bright red bands withnonglycosylated proteins more faintly stained. Using a 520 nm band passfilter revealed that the green signal from Compound 5 had diminishedsubstantially. Analysis of the gel on a laser gel scanner, such as theFuji FLA-3000 (Fuji Film Co., Tokyo, Japan), using the 532 nm secondharmonic generation laser revealed that the proteins were fairlyuniformly labeled with SYPRO RED dye. Without wishing to be bound bytheory, this example illustrates that Compound 5 absorbs UV radiationand transfers it to SYPRO RED dye where it is emitted at a substantiallylonger wavelength (640 nm). This increases the effective Stoke's shiftof the glycoprotein detection system from 245 to 350 nm.

EXAMPLE 33 Edman Sequencing of Glycoproteins Electroblotted to TransferMembranes

Glycoproteins of interest are subjected to electrophoresis, subsequentlytransferred to poly (vinylidene difluoride) membrane and stained asdescribed in Example 27. After target proteins are identified, the bandsare excised with a sharp razor. For internal protein sequencing, thetarget proteins are excised from the membrane, subjected to in-situproteolytic cleavage, for 3 hours at 37° C., and in the presence of 10%acetonitrile, 3% Tween-80 in 100 mM NH₄HCO₃, pH 8.3. Resulting fragmentsare then separated by micro-bore reverse phase HPLC. Selected peakfractions are analyzed by automated Edman degradation. Glycoproteinssubjected to Edman sequencing are expected to produce high qualityspectra with excellent initial and repetitive sequencing yields.

EXAMPLE 34 Matrix-Assisted Laser Desorption Mass Spectrometry-BasedIdentification of Glycoproteins Electroblotted to Transfer Membranes

Glycoproteins of interest were subjected to electrophoresis,subsequently transferred to poly (vinylidene difluoride) membrane andstained as described in Example 27. After target proteins wereidentified, the bands were excised with a sharp razor. Bands were thenwashed 3 times 5 minutes in 25 mM ammonium bicarbonate pH 7.8, 10%methanol and allowed to dry. After drying, the bands were cut into 1-2mm squares and incubated in 20 μg/mL trypsin in digestion buffer (25 mMammonium bicarbonate, pH 7.8 with 1% octyl β-glucoside and 10% methanoladded). Sufficient volume of the trypsin digestion mixture was added tocover the membrane squares. Proteins were digested at room temperaturefor 5-6 h and then incubated overnight at 27-28° C. The peptides wereextracted with formic acid:ethanol (1:1), and then lyophilized. Afterlyophilization, the peptides were resuspended in water for analysis bymatrix assisted laser desorption ionization mass spectrometry(MALDI-MS). Equal volumes of the peptide digests were mixed withα-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 70%acetonitrile/H₂O). The mixture was spotted onto the sample plate and airdried prior to analysis. MALDI-MS analysis was performed in a VoyagerMass Spectrometer, (PerSeptive BioSystems, Framingham, Mass., USA). Theinstrument was calibrated with Substance-P (1347.7 Da) and insulin(5731.4 Da). The peptide masses obtained from the trypsinized proteinwere used to search the EMBL peptide database using the PeptideSearchengine available on the world wide web, (www.mann.emblheidelberg.de).Proteins were expected to be readily identified with good peptidesequence coverage.

EXAMPLE 35 Oligosaccharide Profiling

A glycoprotein, such as α1 acid-glycoprotein (50-500 μg), is prepared in5 μL of 1% SDS, 0.5 M 2-mercaptoethanol, 0.1 M EDTA and incubated atroom temperature for 30 minutes. The sample is then diluted with 40 μLof 0.2 mM sodium phosphate buffer, pH 8.6 and mixed thoroughly. Thesample is heated in a boiling water bath for 5 minutes and then allowedto cool to room temperature. 5 μL of 7.5% Nonidet P-40 is added, thesample is mixed and 5 μL PNGase F (1.0 unit) is added. The tube is mixedthoroughly and then incubated at 37° C. for 20 hours. Then, 165 μL coldethanol is added and the sample is incubated on ice for at least 1 hour.The sample is centrifuged at 10,000 g for 2 minutes at room temperature.The supernate, containing the released glycans is collected and driedusing a centrifugal vacuum evaporator. The dried glycans are suspendedin 20 μL 5 μM Compound 35, 3% acetic acid, 2% dimethylsulfoxide andincubated for 16 hours at 37° C. Sodium cyanoborohydride may optionallybe added to this reaction mixture. Dry the sample mixture in acentrifugal vacuum evaporator for about 1 hour. Though some heating maybe required to vaporize the DMSO, temperatures greater than 45° C.should be avoided. The dried sample is dissolved in glycerol-water (1:4,v/v). The derivatized oligosaccharides are separated on a 14 cm long 30%T, 2.6% C polyacrylamide gel and electrophoresis is performed usingstandard protocols, omitting SDS from the gels and buffers. Sulfonatedversions of the reagents of the invention are preferred for thisapplication as they confer a negative charge to neutral and weaklycharged glycans. Tris-borate gel buffer systems allow unsulfonatedreagents to be used for the detection of neutral glyeans, however, ascommonly used with 2-aminoacridone-derivatized glycans. Afterelectrophoresis, green fluorescent bands may be visualized by eye, byphotography or by CCD-camera-based imaging.

EXAMPLE 36 Cytochemical Detection of DNA in Cells

MRC-5 human lung fibroblasts, NIH-3T3 murine fibroblasts, and bovinepulmonary artery endothelial cells were removed from culture and washedtwice with warm phosphate-buffered saline (PBS) at pH 7.4. Cells werethen fixed with cold 100% ethanol at −20° C. for 10 minutes, washedtwice with PBS, and incubated in 5 N hydrochloric acid for 30 minutes at25° C. The samples were then washed twice in PBS and incubated in 5 μMCompound 5, 3% acetic acid, 2% dimethylformamide, 0.25 M magnesiumchloride for 120 minutes at 25° C., protected from light. Samples werewashed twice in 3% acetic acid and mounted in a glycerol-based mountingmedium before being sealed with paraffin wax for observation.

Fluorescence microscopy was performed using a Nikon Eclipse 800 uprightmicroscope equipped with a 100 watt mercury light source. Visualizationof the fluorescent signal was accomplished using filters with anexcitation wavelength between 320-400 nm and an emission filter withwavelengths between 500-560 nm. Images were acquired using a MicroMax1300 YHS, 12 bit digital camera (Princeton Instruments, Inc.),controlled using MetaMorph software (Universal Imaging Corp).Microscopic observation revealed distinct nuclear green fluorescentsignal in acid-treated cells and no signal in untreated samples. At highmagnification, punctate cytoplasmic signal was observed which mayrepresent mitochondrial DNA.

EXAMPLE 37 Cytochemical Detection of Polysaccharides in Plastic-EmbeddedTissue

Aldehyde blockage is performed using chlorous acid as described inExample 36. Alternatively, thiosemicarbazide blockage (1 mg/mLmethylcellosolve: acetic acid, 95:5) may be employed instead. In thiscase the sample is incubated for 30 minutes at 25° C. in the blockingsolution. The sample is then rinsed in distilled water for 10 minutes,then treated with 1% periodic acid in water for 10 minutes. The sampleis washed in distilled water for 5 minutes and then stained in 5 μMCompound 5, 3% acetic acid, 2% dimethylformamide, and 0.25 M magnesiumchloride for 15-20 minutes. The sample is then rinsed in distilled waterfor 5 minutes, blow-dried and mounted using standard methods.Polysaccharides such as glycogen are observed to be bright green byfluorescence microscopy.

EXAMPLE 38 Preparation of a Reagent-Dextran Conjugate

A solution of carboxymethyldextran (average MW 70,000) havingapproximately 20 carboxylic acid groups is prepared by dissolved thedextran in water whose pH has been adjusted to 6.0 to a finalconcentration of 10 mg/mL. The solution resulting is treated with 5equivalents of ethyldimethylaminopropylcarbodiimide (EDAC) for 15minutes at room temperature with stirring. To the solution is then added1 equivalent of Compound 5, and the reaction mixture is stirredovernight. The solution is then poured into ethanol, and the resultingprecipitate is separated from unconjugated Compound 5 by extensivedialysis against pH 7 phosphate buffer, then water. The solution ofconjugated dextran is lyophilized to yield a green-fluorescent solidthat should be useful as a fluorescent tracer.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, numerousmodifications, substitutions, and alterations are possible withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, numerousmodifications, substitutions, and alterations are possible withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1. A compound of the formula:

wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R⁶ is -L-Z; L isarylene, or a C₁₋₆ perfluoroalkylene; and Z is a (C═0)NR⁸NH₂ (carbonylhydrazide), —NR⁶—NH₂ (hydrazide), —(SO₂)NR⁶NH₂ (sulfonyl hydrazide) or—(C═S)NR⁶NH₂ (thiocarbonyl hydrazide); R¹¹-R¹⁴ are independently H, C₁₋₆alkyl, C₁₋₆ alkoxy, C₁₋₆ perfluoroalkyl, C₁₋₆ alklyamino,di(C₂₋₁₂-alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro,phenyl, or sulfo; R²¹-R²⁴ are independently H, C₁₋₆ alkyl, C₁₋₆ alkoxy,C₁₋₆ perfluoroalkyl, C₁₋₆ alklyamino, di(C₂₋₁₂alkyl)amino, amino,carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z; R6 is Hor alkyl having 1-6 carbons.
 2. The compound, as claimed in claim 1wherein Q is carbonyl.
 3. The compound, as claimed in claim 1, wherein Lis arylene.
 4. The compound, as claimed in claim 1, wherein R¹¹-R¹⁴ areeach H.
 5. The compound according to claim 1, wherein said compound isselected from the group consisting of


6. The compound according to claim 1, wherein said Z is sulfonylhydrazide or carbonyl hydrazide.
 7. The compound according to claim 1,wherein L is C₁₋₆ perfluoroalkylene.
 8. The compound according to claim1, wherein R²¹-R²⁴ are each hydrogen.
 9. A compound of the formula:

wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R⁶ is -L-Z; L is asingle covalent bond; Z is —NR⁶—NH₂ (hydrazide); R¹¹-R¹⁴ areindependently H, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ perfluoroalkyl, C₁₋₆alklyamino, di(C₂₋₁₂-alkyl)amino, amino, carboxy, cyano, halogen,hydroxy, nitro, phenyl, or sulfo; and R²¹-R²⁴ are independently H, C₁₋₆alkyl, C₁₋₆ alkoxy, C₁₋₆ perfluoroalkyl, C₁₋₆ alklyamino,di(C₂₋₁₂-alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro,phenyl, sulfo, or -L-Z; R6 is H or alkyl having 1-6 carbons.